1. Field of the Invention
The present invention relates to the use of ozone in the activation of the surface of a poly(cyclic olefin) substrate in the absence of UV light, for the production of a substrate suitable for a fluorescence-based assay.
2. Background Art
High throughput technologies, particularly microarrays, have had a large impact in life sciences. Microarrays not only permit carrying out a large number of experiments in a parallel format, but also decrease significantly the amounts of reagents required for the analysis. Traditionally, glass has been the substrate of choice for the fabrications of microarrays and this is partly due to the existence of rather simple and well-known surface modification methods that are based on silane chemistry.
The main problem with the use of glass is expense. Chemically modified glass slides for microarray technologies are expensive and therefore plastic slides are an economical alternative. Moreover, micromachining of glass is an extremely expensive process and as a consequence this material is not suitable for 1) enhancing microarray slides with micro/nanostructures which would increase the surface area and therefore enhance the detection limit and 2) fabrication of nano/microfluidic-based systems for high throughput screening devices.
Several approaches are presented in the literature for the chemical modification of a poly(cyclic olefin) substrate. For instance, plasma treatment has been employed successfully to increase the hydrophilicity of the plastics (J. Kai, Y.-S. Sohn, C. H. Ahn, Micro Total Analysis Systems 2002, Proceedings of the mTAS 2002 Symposium, 6th, Nara, Japan, Nov. 3-7, 2002 2002, 1, 419-421; Y.-S. Sohn, J. Kai, C. H. Ahn, Sens. Lett. 2004, 2, 3, 4, 171-174; D. Nikolova, E. Dayss, G. Leps, A. Wutzler, Surf. Interface Anal. 2004, 36, 8, 689-693; A. Puntambekar, S. Murugesan, R. Trichur, H. J. Cho, S. Kim, J.-W. Choi, G. Beaucage, C. H. Ahn, Micro Total Analysis Systems 2002, Proceedings of the mTAS 2002 Symposium, 6th, Nara, Japan, Nov. 3-7, 2002 2002, 1, 425-427; C. Ahn, S. Kim, H. Chao, S. Murugesan, G. Beaucage, Materials Research Society Symposium Proceedings 2002, 729, BioMEMS and Bionanotechnology, 131-136). This is an aggressive method that generates a large number of polar groups such as hydroxyl groups. However, this technique is difficult to implement on an industrial scale since it requires expensive high vacuum systems.
A second approach involves reacting the poly(cyclic olefin) surface with highly reactive intermediates (H. J. Mathieu, Y. Chevolot, L. Ruiz-Talor, D. Leonard, Adv. Polym. Sci. 2003, 162, 1-34; J. J. Tate, J. Persinger, B. Bartholomew, Nucleic Acids Res. 1998, 26, 6, 1421-1426), such as free radicals (T. Rohr, D. F. Ogletree, F. Svec, J. M. J. Frechet, Adv. Funct. Mater. 2003, 13, 4, 264-270), nitrenes (K. A. Schnapp, R. Poe, E. Leyva, N. Soundararajan, M. S. Platz, Bioconjug. Chem. 1993, 4, 172-177; K. A. Schnapp, M. S. Platz, Bioconjug. Chem. 1993, 4, 178-183; J. F. W. Keana, S. X. Cai, J. Org. Chem. 1990, 55, 3640-3647) or carbenes (W. Kramer, S. Schneider, J. Lipid Res. 1989, 30, 1281-1288; K. Bergmann, K. E. Carlson, J. A. Katzenellenbongen, Bioconjug. Chem. 1994, 5, 141-150; A. Collioud, J.-F. Clemence, M. Sanger, H. Sigrist, Bioconjug. Chem. 1993, 4, 528-536). Although several degrees of success have been achieved with this approach, some of the precursors for the reactive intermediates are expensive (e.g., fluorinated aryl azides). Moreover, this approach results in many cases in inhomogenous surfaces that are not useful for assay applications (L. M. Shamansky, C. B. Davis, J. K. Stuart, W. G. Kuhr, Talanta 2001, 55, 909-918).
Ozone treatment of poly(cyclic olefins) has been used with respect to preparing a surface for adhesion to other materials on a macroscopic scale. EP 694,567 A2 and EP 694,568 A2 disclose ozone treatment of poly(cyclic olefins) with a view to producing a material that has good adherence properties to metal, glass or plastic. The ozone is preferably produced with a UV source, consistent with aggressive oxidation suitable for that application. US Patent Application 20050181531, filed Jan. 26, 2005, discloses use of ozone-treated poly(cyclic olefin) to improve adhesiveness of a base layer in coating with a bright decoration layer for use in a radar apparatus. Again, the surface of the poly(cyclic olefin) was aggressively treated with plasma ion etching and/or highly concentrated ozone water with a view to subsequent macroscopic modifications.